Alzheimer's disease (AD) is characterized by progressive impairment of cognitive function which are often accompanied by emotional and sleep disturbances. These symptoms are the result of progressive degeneration of synapses and death of neurons in brain regions involved in learning and memory and stress responses including the hippocampus, entorhinal cortex and basal forebrain. AD patients exhibit extensive deposition of aggregates of a protein called amyloid beta-peptide that forms ?plaques? which are typically associated with the neuronal pathology. Most cases of AD are sporadic with no clear genetic basis, whereas a small percentage of cases are caused by mutations in one of three genes: the amyloid precursor protein (APP), presenilin-1 (PS1) or presenilin-2 (PS2). This laboratory has developed cell culture and mouse models of AD, and have used these models to elucidate the biochemical and molecular events responsible for neuronal dysfunction and death in AD. Our previous studies provided evidence that the neurodegenerative process in AD may be triggered by age-related increases in oxidative and metabolic stress in combination with the deposition of amyloid beta-peptide. Below is a summary of progress in elucidating the molecular and cellular abnormalities in AD, and in identifying novel preventative and therapeutic approaches for this devastating disorder. Pathogenic Mechanism of Presenilin-1 Mutations: Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. PS1 mutations may perturb cellular Ca(2+) homeostasis and thereby render neurons vulnerable to excitotoxicity and apoptosis. We found that PC12 cells expressing PS1 mutations and primary hippocampal neurons from PS1 mutant knockin mice exhibit greatly increased levels of ryanodine receptors (RyR) and enhanced Ca(2+) release following stimulation with caffeine. Double-labeling immunostaining and co-immunoprecipitation analyses indicate that PS1 and RyR are colocalized and interact physically. Caffeine treatment sensitizes neurons expressing mutant PS1 to apoptosis induced by amyloid beta-peptide, a neurotic peptide linked to the pathogenesis of AD. When taken together with recent evidence for alterations in RyR in brains of AD patients, our data suggest that PS1 mutations may promote neuronal degeneration in AD by increasing transcription and translation of RyR and altering functional properties of ryanodine-sensitive Ca(2+) pools. To further elucidate the cellular mechanisms underlying these disturbances, we studied calcium signaling in fibroblasts isolated from mutant PS1 knockin mice. Mutant PS1 knockin cells exhibited a marked potentiation in the amplitude of calcium transients evoked by agonist stimulation. These cells also showed significant impairments in capacitative calcium entry (CCE, also known as store-operated calcium entry), an important cellular signaling pathway wherein depletion of intracellular calcium stores triggers influx of extracellular calcium into the cytosol. Notably, deficits in CCE were evident after agonist stimulation, but not if intracellular calcium stores were completely depleted with thapsigargin. Treatment with ionomycin and thapsigargin revealed that calcium levels within the ER were significantly increased in mutant PS1 knockin cells. Collectively, our findings suggest that the overfilling of calcium stores represents the fundamental cellular defect underlying the alterations in calcium signaling conferred by presenilin mutations. Homocysteine Endangers Neurons: Elevated plasma levels of the sulfur-containing amino acid homocysteine increase the risk for atherosclerosis, stroke, and possibly Alzheimer's disease, but the underlying mechanisms are unknown. We have discovered that homocysteine apoptosis in rat hippocampal neurons. DNA strand breaks and associated activation of poly-ADP-ribose polymerase (PARP) and NAD depletion occur rapidly after exposure to homocysteine and precede mitochondrial dysfunction, oxidative stress, and caspase activation. The PARP inhibitor 3-aminobenzamide (3AB) protects neurons against homocysteine-induced NAD depletion, loss of mitochondrial transmembrane potential, and cell death, demonstrating a requirement for PARP activation and/or NAD depletion in homocysteine-induced apoptosis. Caspase inhibition accelerates the loss of mitochondrial potential and shifts the mode of cell death to necrosis; inhibition of PARP with 3AB attenuates this effect of caspase inhibition. Homocysteine markedly increases the vulnerability of hippocampal neurons to excitotoxic and oxidative injury in cell culture and in vivo, suggesting a mechanism by which homocysteine may contribute to the pathogenesis of neurodegenerative disorders. Protection Against Amyloid Toxicity by Manipulation of Glucose Metabolism: Mild metabolic stress can increase resistance of neurons in the brain to subsequent more severe insults, as exemplified by the beneficial effects of heat shock and ischemic preconditioning. Studies of Alzheimer's disease and other age-related neurodegenerative disorders indicate that dysfunction and degeneration of synapses occur early in the cell death process, and that oxidative stress and mitochondrial dysfunction are central events in this pathological process. We showed that administration of 2-deoxy-D-glucose (2DG), a nonmetabolizable glucose analog that induces metabolic stress, to rats and mice can increase resistance of neurons in the brain to excitotoxic, ischemic, and oxidative injury. We further showed that administration of 2DG to adult rats (daily i.p. injections of 100 mg/kg body weight) increases resistance of synaptic terminals to dysfunction and degeneration induced by amyloid beta-peptide and ferrous iron, an oxidative insult. The magnitude of impairment of glucose and glutamate transport induced by amyloid beta-peptide and iron was significantly reduced in cortical synaptosomes from 2DG-treated rats compared to saline-treated control rats. Mitochondrial dysfunction, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated after exposure to amyloid beta-peptide and iron in synaptosomes from 2DG-treated rats. Levels of the stress proteins HSP-70 and GRP-78 were increased in synaptosomes from 2DG-treated rats, suggesting a mechanism whereby 2DG protects synaptic terminals. We conclude that 2DG bolsters cytoprotective mechanisms within synaptic terminals, suggesting novel preventative and therapeutic approaches for neurodegenerative disorders. Development of Drugs to Prevent Neuronal Death in Alzheimer's Disease: The tumor suppressor protein p53 is essential for neuronal death in several experimental settings and may participate in human neurodegenerative disorders. Based upon recent studies characterizing chemical inhibitors of p53 in preclinical studies in the cancer therapy field, we synthesized the compound pifithrin-alpha and evaluated its potential neuroprotective properties in experimental models relevant to the pathogenesis of stroke and neurodegenerative disorders. Pifithrin-alpha protected neurons against apoptosis induced by DNA-damaging agents, amyloid beta-peptide and glutamate. Protection by pifithrin-alpha was correlated with decreased p53 DNA-binding activity, decreased expression of the p53 target gene BAX and suppression of mitochondrial dysfunction and caspase activation. Mice given pifithrin-alpha exhibited increased resistance of cortical and striatal neurons to focal ischemic injury and of hippocampal neurons to excitotoxic damage. These preclinical studies demonstrate the efficacy of a p53 inhibitor in models of Alzheimer's disease and stroke, and suggest that drugs that inhibit p53 may reduce the extent of brain damage in related human p